Reductive nitrosylation of ferric cyanide horse heart myoglobin is limited by cyanide dissociation
Reductive nitrosylation of ferric cyanide horse heart myoglobin is limited by cyanide dissociation
复制标题
DOI：
10.1016/j.bbrc.2010.01.092
复制
发表时间：
2010-03-05
影响因子：
3.1
通讯作者：
Coletta, Massimo
中科院分区：
文献类型：
--
作者：
Ascenzi, Paolo;di Masi, Alessandra;Coletta, Massimo
氰化物以非常高的亲和力与铁血红素蛋白结合，反映了非常低的解离速率常数 (k(off))。由于没有技术可用于估计 k(off)，我们在此报告一种基于捕获铁肌红蛋白 (Mb(III)) 的不可逆还原亚硝基化反应来确定 k(off) 的方法。在 pH 9.2 和 20.0 摄氏度下测定氰化铁马心肌红蛋白（Mb(III)-氰化物）中氰化物解离的 k(off) 值。混合 Mb(III)-氰化物和 NO 溶液会导致吸收光谱变化，反映 Mb(III)-氰化物消失并同时形成亚硝基化 Mb。由于 Mb(III) 还原亚硝基化的动力学比 Mb(III)-氰化物解离快得多，因此可以直接确定代表限速步骤的 k(off) 值。实验获得的 k(off) 值与氰化物与 Mb(III) 结合的二阶速率常数 (k(on)) 和解离平衡常数 (K) 值计算得出的值非常匹配 (k(off) = k(on) x K）。 (C) 2010 Elsevier Inc. 保留所有权利。
Cyanide binds to ferric heme-proteins with a very high affinity, reflecting the very low dissociation rate constant (k(off)). Since no techniques are available to estimate k(off), we report herewith a method to determine k(off) based on the irreversible reductive nitrosylation reaction to trap ferric myoglobin (Mb(III)). The k(off) value for cyanide dissociation from ferric cyanide horse heart myoglobin (Mb(III)-cyanide) was determined at pH 9.2 and 20.0 degrees C. Mixing Mb(III)-cyanide and NO solutions brings about absorption spectral changes reflecting the disappearance of Mb(III)-cyanide with the concomitant formation of ferrous nitrosylated Mb. Since kinetics of reductive nitrosylation of Mb(III) is much faster than Mb(III)-cyanide dissociation, the k(off) value, representing the rate-limiting step, can be directly determined. The k(off) value obtained experimentally matches very well to that calculated from values of the second-order rate constant (k(on)) and of the dissociation equilibrium constant (K) for cyanide binding to Mb(III) (k(off) = k(on) x K). (C) 2010 Elsevier Inc. All rights reserved.